Born in Cleveland, Ohio, Jason Dufner moved to the Washington, D.C. area when he was 11 years old, and then to Fort Lauderdale, Fla., when he was 14. It was there that he started playing golf, and played for St. Thomas Aquinas High School during his sophomore, junior and senior years.

In his early professional career, Dufner struggled to hold down a place on the PGA Tour. He was a member of the PGA Tour in 2004 and the Nationwide Tour in 2001, 2002, 2003, 2005 and 2006. Dufner won two events during his time on the Nationwide Tour, the BUY.COM Wichita Open in 2001 and the LaSalle Bank Open in 2006. He finished in 8th place on the Nationwide Tour money list in 2006 to earn his PGA Tour card for 2007. He finished 127th in the FedEx Cup standings in 2007, and failed to qualify at the PGA Tour Q-School, where he finished T149. He retained conditional status for the 2008 PGA Tour season, and then finished T11 at the Q-School that year to earn his card for 2009. He has been a member of the PGA Tour every year since.

At the PGA Championship in August, Dufner won his maiden major championship with a two-stroke victory at Oak Hill Country Club in Rochester, New York. After beginning the tournament with a two-under round of 68, Dufner tied the record for all-time lowest round in a major and broke the course record at Oak Hill Country Club by shooting a seven-under-par 63 in the second round. He carded five birdies and an eagle, the latter achieved when he holed out from 105 yards on the par-4 second hole. Dufner had the chance to finish with a 62 and the outright lowest round ever in a major, but he left his 12-foot putt for birdie at the 18th hole short. The record-breaking round took Dufner into a two-shot lead going into the weekend. In round three, Dufner shot a one-over round of 71 to fall out of the lead, one stroke behind Jim Furyk. That marked the second time in three years that Dufner was in the final group of the PGA Championship on Sunday.

In the final round, Dufner started well by making a birdie at the 4th hole, which took him into a share of the lead alongside Furyk. Further birdies at the 5th and 8th holes, allied with Furyk bogeying the 9th, opened up a two-stroke advantage for Dufner entering the back nine. He missed a birdie putt on the 10th which would have moved him three strokes ahead and then holed a short putt on the 11th which almost lipped out for par. Having turned into a two-man race, both Dufner and Furyk made four straight pars between the 12th and 15th holes. On the 16th, Dufner stiffed his approach to within 18 inches, but Furyk holed a lengthy birdie putt to stay in touch. After both players bogeyed the 17th, Dufner held a two-stroke lead going up the final hole. Both players found the rough with their drives and approach shots and after Furyk could only manage a bogey, Dufner lagged his par putt down to the hole to tap in, finishing 10 under par for the tournament and a two-stroke victory. Dufner moved back inside the top ten, at 8th in the rankings, after the victory.

Dufner qualified for his first Ryder Cup team in 2012, finishing with a record of 3–1, including a 2-up win over Peter Hanson in the singles competition.

Dufner's laid-back mannerisms have led to the phrase "Dufnering," which specifically refers to a slumped sitting position with an expressionless face. He married his longtime girlfriend, Amanda Boyd, on May 5, 2012.